| description |
Chromatin transactions are typically studied in vivo, or in vitro using artificial chromatin lacking the epigenetic complexity of the natural material. Attempting to bridge the gap between these approaches, we established a system for isolating the yeast genome as a library of mono-nucleosomes harbouring the natural epigenetic signature, suitable for biochemical manipulation. Combined with deep sequencing, this library was used to investigate the stability of individual nucleosomes, and – as proof of principle - the nucleosome preference of the chromatin remodeling complex, RSC. In order to generate a library of native yeast nucleosomes, we developed a three-step purification protocol: first, purified yeast nuclei were incubated with micrococcal nuclease (MNase), which preferentially digests naked DNA to generate short chromatin fragments. The resulting fragments were extracted from the nuclei, then bound to and eluted from DEAE sepharose. This was followed by ultracentrifugation through a sucrose gradient to separate the fragments by length to further remove contaminating proteins and free DNA. We chose a simple disassembly assay, which involves incubating the nucleosome library with ATP and the histone chaperone Nap1, with or without RSC. In this assay, RSC binds to nucleosomes and transfers the histones to Nap1, thereby releasing ‘naked’ DNA. Under certain conditions, reaction intermediates can be observed (tetramers or hexasomes), but for simplicity we chose to compare the input nucleosomes with the final naked DNA product. To separate the RSC-dependent released DNA from the non-remodeled nucleosomes, the reactions were subjected to native agarose gel electrophoresis, and DNA of the four bands isolated by gel-extraction. The upper bands, harboring nucleosomes, were named NUC (no RSC) and NUCR (with RSC), whereas the lower, ‘naked’ DNA bands were named DNA (no RSC) and DNAR (with RSC). |